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Thirty boys and thirty girls aged 10 to 12 years from Huaibei primary school of Beijing (A boarding school for nine-year compulsory education, where our research can be carried out to keep track of the children and avoid the loss of samples due to higher education) were recruited to participate in this study. All of the children were subjected to a physical examination and were considered to be healthy and prepubertal if no evidence of menarche (girls) or secondary sexual characteristics (boys) was found. Three girls withdrew because of diarrhea in the pre-experiment stage. The trial was registered at the Chinese Clinical Trial Registry (No:ChiCTR-OCH-14004302) and approved by the Ethical Committee of the National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention. Informed consent was obtained and provided by at least one parent of each participant.
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A stable iron isotope enriched for 57Fe was obtained in metal form from Trace Sciences International Corporation (54Fe: 0.0023% ± 0.0008%, 56Fe: 1.27% ± 0.06%, 57Fe: 96.610% ± 0.008%, and 58Fe: 2.11% ± 0.05%, as determined by the National Institute of Metrology) and used to prepare a ferrous sulfate solution by the addition of 0.5 mol/L sulfuric acid until the metal was completely dissolved[13]. After dilution, vitamin C was added to eliminate the unpleasant flavor caused by the ferrous sulfate and to maintain an acidic environment. The oral solution was prepared according to a previously established method[14]. The solution complied with the hygienic standards of the China National Center for Food Safety Risk Assessment and was sealed and stored in a cool and dark environment until use.
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The study flow chart is shown in Figure 1. At the first 14 d, to achieve the highest possible reduction in the saturation of mucosal cells with iron, the subjects were assigned to a low-iron diet during the trial and not allowed to consume food or drink other than those specified and water, the actual intake of food served for the subjects every day was recorded. Thirty minutes before breakfast and dinner, the subjects were orally administered 3 mg of 57Fe in the form of the ferrous sulfate solution, and this process was repeated to obtain a total administration of 57Fe of 30 mg over a 5 d period. The exact 57Fe intake for each subject was calculated by the 57Fe abundance in the solution and food and the amount of intake.
Following an overnight fast, 10 mL of blood was obtained from each participant in 0 day, 14th day, 28th day, 60th day, and 90th day after isotope administration. The RBCs in the blood samples were divided into two parts: 5 mL of the collected blood was subjected to common testing, such as analyses of the hemoglobin (Hb) and serum ferritin (SF) contents, as described previously[3], and the other 5 mL of blood was analyzed by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) to obtain the isotope ratio, as detailed by von Blanckenburg et al.[15].
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The total amount of 57Fe incorporated into erythrocytes (57Feinc) was calculated according to the following formula[16]:
$$ ^{57}{\text{Fe}}{_{\text{inc}}} = \frac{{{{MIR}}_{{{57/56}}}^{{t}}-{{MIR}}_{{{57/56}}}^{{0}}}}{{{{MIR}}_{{\bf{57/56}}}^{{0}}}} \times {{F}}{{{e}}_{{{circ}}}} \times {{0}}{{.0214}} $$ where 57Feinc is expressed in mg,
${{{MIR}}_{{\bf{57/56}}}^{{t}}}$ is the 57Fe/56Fe ratio at the specified time after isotope administration,${{{MIR}}_{{\bf{57/56}}}^{\bf{0}}}$ is the 57Fe/56Fe ratio at baseline, and Fecirc is the total amount of iron in the circulation at the specified time, which is expressed in mg and estimated as follows[17]:$$ \text{Fe}{_{\text{circ}}} = \text{BV} \times \text{Hb} \times 3.47 $$ where 3.47 is the concentration of iron in Hb expressed in mg/g, Hb is the hemoglobin concentration (g/L), and BV is the blood volume expressed in mL. In this study, BV was estimated by the sex and the body weight (BW), as described by Etcheverry et al.[18], as follows:
$$ \text{For boys: BV}= \text{0.075 3} \times \text{BW (kg)}-\text{0.05} $$ $$ \text{For girls: BV} = \text{0.075 3} \times \text{BW (kg)} + 0.01 $$ Thus, the incorporation of 57Fe into erythrocytes was calculated as:
$$\frac{{{}^{{{57}}}{{Fe}}_{{{inc}}}}}{{{}^{{{57}}}{{Fe}}\;{{given}}\left( {{{oral}}} \right)}}$$ -
The results are presented as the mean values ± standard deviation (SD). Repeated measures ANOVA was used for comparisons between groups. All of the statistical analyses were performed using the Statistical Product and Service Solutions 13.0 software.
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Anthropometric measurements of the subjects are described in Table 1. There were no differences in the average age, height, sitting height, body weight, RBC, Hb and SF. According to the standard of iron deficiency which was defined as serum ferritin below 14 μg/L[19], the iron status of all of the children was good.
Table 1. Baseline data of the children according to sex (mean ± SD)
Characteristics Boys (n = 30) Girls (n = 27) Age (years) 10.6 ± 0.2 10.4 ± 0.2 Height (cm) 146.2 ± 7.5 144.9 ± 6.5 Sitting height (cm) 78.1 ± 4.3 77.0 ± 3.7 Body weight (kg) 41.5 ± 12.0 37.8 ± 6.9 Body mass index (kg/cm2) 19.2 ± 4.4 17.9 ± 2.6 Red blood cells (× 1012/L) 4.4 ± 0.2 4.5 ± 0.3 Hemoglobin (g/L) 133.1 ± 4.9 130.8 ± 6.1 Serum ferritin (μg/L) 57.67 ± 6.65 41.08 ± 4.96 -
The total energy intakes were slightly higher than the RNI (Table 2). According to the RNI[20], the energy proportions from carbohydrates, proteins and fats are all reasonable, and the intakes of iron in boys was slightly higher than that in girls (P< 0.05).
Table 2. Intake of main dietary nutrients according to sex (mean ± SD)
Characteristic Boys (n = 30) Girls (n = 27) Actual intake (g/d) Energy (%) Actual intake (g/d) Energy (%) Carbohydrate 352.7 ± 33.9 56.3 ± 5.2 294.2 ± 18.5 58.4 ± 4.0 Protein 75.5 ± 4.1 15.8 ± 3.1 69.3 ± 6.2 13.9 ± 2.9 Fat 52.5 ± 8.4 31.4 ± 4.7 45.6 ± 6.4 29.5 ± 2.4 Iron 8.1 ± 1.1* − 7.3 ± 0.9* − Note. −: nor measured; *: (mg/d). -
The mean 57Fe/56Fe ratios increased starting 14th day, reached a peak at 60 d and then decreased. The percentage of the administered dose of 57Fe that was incorporated into erythrocytes ranged from 13% to 27%. The erythrocyte incorporation of 57Fe presented a trend consisting of an initial increase to a peak at 60 d; the mean peak values were 19.67% and 21.33%. As shown in Table 3 and Figure 2, the erythrocyte incorporation of 57Fe of the boys was significantly lower than that of the girls in 60th day (F = 43.842, P < 0.0001), but no difference was observed in 14th day, 28th day, and 90th day ( F = 2.05, P = 0.157). There was an interaction between gender and time point both in 57Feinc and erythrocyte incorporation of 57Fe (F = 31.45, 32.16, P < 0.0001), and erythrocyte 57Fe incorporation in girls was significantly difference at each time point, as calculated by repeat measure ANOVA (P < 0.05).
Figure 2. Trend obtained for the incorporation of 57Fe into erythrocytes over time (of dose) in prepubertal children according to sex.
Table 3. Erythrocyte 57Fe incorporation over time according to sex (mean ± SD)
Time (d) MIRt 57/56 57Feinc (mg) Erythrocyte incorporation of 57Fe
(percentage of dose, %)Boys (n = 30) 14th 0.026415 ± 0.000831 5.16 ± 0.75 18.79 ± 0.47 28th 0.026649 ± 0.001035 5.27 ± 0.77 19.20 ± 0.62# 60th 0.026987 ± 0.001142 5.40 ± 0.62 19.67 ± 0.56#* 90th 0.026698 ± 0.002414 5.32 ± 0.74 19.36 ± 0.44#*@ Girls (n = 27) 14th 0.026935 ± 0.001450 5.32 ± 0.88 19.49 ± 0.47 28th 0.027237 ± 0.001470 5.51 ± 0.66 20.19 ± 0.85# 60th 0.027750 ± 0.001336 5.82 ± 0.43 21.33 ± 0.59#* 90th 0.027391 ± 0.001273 5.60 ± 0.62 20.52 ± 0.68#*@ Note. #The mean value is significantly different from that obtained at 14 d (P < 0.0001); *The mean value is significantly different from that obtained at 28 d (P < 0.0001); @The mean value is significantly different from that obtained at 28 d (P < 0.0001).
doi: 10.3967/bes2020.056
Evaluation of Erythrocyte Iron Incorporation in Beijing Prepubertal Children Using a Single Stable Isotope Tracer Method
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Abstract:
Objective To analyze the rate of erythrocyte iron incorporation and provided guidance for the iron nutrition for prepubertal children. Methods Fifty-seven prepubertal children of Beijing were involved in this study and each subject was orally administered 3 mg of 57Fe twice daily to obtain a total of 30 mg 57Fe after a 5-d period. The stable isotope ratios in RBCs were determined in 14th day, 28th day, 60th day, and 90th day. The erythrocyte incorporation rate in children was calculated using the stable isotope ratios, blood volume and body iron mass. Results The percentage of erythrocyte 57Fe incorporation increased starting 14 th day, reached a peak at 60 d (boys: 19.67% ± 0.56%, girls: 21.33% ± 0.59%) and then decreased. The erythrocyte incorporation rates of 57Fe obtained for girls in 60th day was significantly higher than those obtained for boys (P < 0.0001). Conclusions The oral administration of 57Fe to children can be used to obtain erythrocyte iron incorporation within 90 d. Prepubertal girls should begin to increase the intake of iron and further studies should pay more attention to the iron status in prepubertal children. -
Table 1. Baseline data of the children according to sex (mean ± SD)
Characteristics Boys (n = 30) Girls (n = 27) Age (years) 10.6 ± 0.2 10.4 ± 0.2 Height (cm) 146.2 ± 7.5 144.9 ± 6.5 Sitting height (cm) 78.1 ± 4.3 77.0 ± 3.7 Body weight (kg) 41.5 ± 12.0 37.8 ± 6.9 Body mass index (kg/cm2) 19.2 ± 4.4 17.9 ± 2.6 Red blood cells (× 1012/L) 4.4 ± 0.2 4.5 ± 0.3 Hemoglobin (g/L) 133.1 ± 4.9 130.8 ± 6.1 Serum ferritin (μg/L) 57.67 ± 6.65 41.08 ± 4.96 Table 2. Intake of main dietary nutrients according to sex (mean ± SD)
Characteristic Boys (n = 30) Girls (n = 27) Actual intake (g/d) Energy (%) Actual intake (g/d) Energy (%) Carbohydrate 352.7 ± 33.9 56.3 ± 5.2 294.2 ± 18.5 58.4 ± 4.0 Protein 75.5 ± 4.1 15.8 ± 3.1 69.3 ± 6.2 13.9 ± 2.9 Fat 52.5 ± 8.4 31.4 ± 4.7 45.6 ± 6.4 29.5 ± 2.4 Iron 8.1 ± 1.1* − 7.3 ± 0.9* − Note. −: nor measured; *: (mg/d). Table 3. Erythrocyte 57Fe incorporation over time according to sex (mean ± SD)
Time (d) MIRt 57/56 57Feinc (mg) Erythrocyte incorporation of 57Fe
(percentage of dose, %)Boys (n = 30) 14th 0.026415 ± 0.000831 5.16 ± 0.75 18.79 ± 0.47 28th 0.026649 ± 0.001035 5.27 ± 0.77 19.20 ± 0.62# 60th 0.026987 ± 0.001142 5.40 ± 0.62 19.67 ± 0.56#* 90th 0.026698 ± 0.002414 5.32 ± 0.74 19.36 ± 0.44#*@ Girls (n = 27) 14th 0.026935 ± 0.001450 5.32 ± 0.88 19.49 ± 0.47 28th 0.027237 ± 0.001470 5.51 ± 0.66 20.19 ± 0.85# 60th 0.027750 ± 0.001336 5.82 ± 0.43 21.33 ± 0.59#* 90th 0.027391 ± 0.001273 5.60 ± 0.62 20.52 ± 0.68#*@ Note. #The mean value is significantly different from that obtained at 14 d (P < 0.0001); *The mean value is significantly different from that obtained at 28 d (P < 0.0001); @The mean value is significantly different from that obtained at 28 d (P < 0.0001). -
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